Project Summary The long-term goal of our research is to understand the principles that permit developmental systems to robustly construct embryos of the correct pattern, shape, and size. Developmental systems face a gamut of variations from different sources including environmental, genetic, and stochastic, which manifest at multiple levels from molecules to cells to organs. In the face of these challenges, organisms have been designed through evolution to buffer the phenotype against these variations in order to robustly achieve a developmental norm, a process Waddington termed canalization. As our knowledge of the molecular and cellular details of patterning systems has expanded, there is now the opportunity to understand the systems level mechanisms that give rise to robust pattern formation. Here we focus on pattern robustness through the lens of scaling and size control. Scaling is a remarkable process in which the size of a pattern can be adjusted to the available size of the tissue. Scaling has fascinated and baffled embryologists since the time of Hans Driesch who in 1885 found that when the blastomeres of a two-cell stage sea urchin embryo are separated, the result is not two partial embryos but rather two complete embryos in which all their pattern is scaled by half. Similar results have since been found in a variety of organisms, but the surgical manipulations required to generate size-reduced animals are generally difficult and result in a lot of variability, thus limiting quantitative investigation. Recently, we have developed a new method for generating zebrafish eggs of different size that is robust and reproducible. Such embryos have qualitatively normal but scaled patterning and can give rise to viable adults. At a molecular level we find that most gene expression patterns (e.g. morphogens and their targets) scale with the tissues they pattern; however, a small subset of genes, the ones that sense tissue size to regulate scaling (e.g. by interacting with morphogens), do not. Thus, these size altered embryos represent a powerful and unique method to identify and determine the mechanisms of pattern scaling. Ultimately, tissue size is determined by balancing the rates of proliferation and differentiation over the course of development. We have found that the balance of proliferation and differentiation in the neural tube is under negative feedback control by mechanical pressure/tissue packing. Here we will use a combination of quantitative imaging, molecular and mechanical perturbations, and computer modeling to determine the systems-level mechanisms that allow: 1) morphogen patterning to scale to fit the available space, and 2) proliferation and differentiation rates to be balanced to cause a tissue to grow to fit the available space. These questions will be addressed in the zebrafish neural tube, but we expect the resulting mechanisms to be widely applicable. Such an integrated understanding is important for diagnosing and treating birth defects such as neural tube defects and in the rational design of engineered tissues.